The pacemaking and conduction system (PCS) of the heart is a profoundly complex structure orchestrating orderly contractions of the cardiac chambers by generating and transmitting action potentials at an appropriate rate, conduction velocity, and incorporating a delay between the chambers to allow for proper ventricular filling. The PCS has been extensively studied in many animal models, however, the clinical relevance of these studies is not entirely clear. In contrast, we propose to conduct these studies in the healthy and diseased human heart in vitro. As evident from developmental studies, the PCS initially forms as an anatomically and functionally continuous structure that is starkly different from working atrial and ventricular myocardium. Subsequently, several distinct cell types develop within the PSC to fulfill the different requirements of pacemaking and conduction properties. The atrio-ventricular junction (AVJ), for example, represents this complexity at its extreme. There is a nearly 100-fold difference in the conduction velocity between the anatomically adjacent compact AV node (~2-3 cm/sec) and the His-Purkinje network (up to 2.5 m/sec). Such a difference is required for the proper delay of excitation between the atria and ventricles, on the one hand, and the synchronized excitation of the ventricles, on the other hand. We hypothesize that this functional heterogeneity has both a structural and molecular basis as the heterogeneity of gene expression encoding gap junctions, ion channels, and receptors of the PCS provides the substrate for normal and abnormal pacemaking and conduction. In this project we will focus on the quantification of the three-dimensional structure and function of the human AVJ. We will use a multimodal biophotonic approach consisting of optical mapping (OM) with voltage-sensitive dye, optical coherence tomography (OCT), and 3D immunohistochemical mapping (IM) of proteins, which define intercellular coupling and cell types. The specific aims of the project are: (1) To investigate molecular, structural and functional mechanisms of the dual pathway AV conduction in the human heart as suggested by opposing protein expression patterns in adjacent anatomic structures;(2) To test the hypothesis that the AV conduction axis may bypass the compact AVN and that can be utilized in resynchronization therapy in hearts with intact slow pathway conduction;(3) To test the hypothesis that an autonomically modulated AVJ pacemaker can replace a sick SAN;(4) To elucidate the basic molecular and structural mechanisms predisposing up to 90,000 patients per year to AVN reentrant tachycardia. The long-term goal of this project is to develop a structure/function framework of the entire human cardiac PCS. During the present funding period we will focus our efforts on the human AVJ. Numerous studies have previously examined the contribution of various isoforms of connexins, ion channels, and receptors to cellular physiology in the cardiac PCS. We, on the other hand, aim to apply a systems physiology approach, to examine factors responsible for the stability of normal pacemaking and conduction, abnormal impulse generation and failed conduction, and arrhythmogenesis mediated by the autonomic nervous system. We will also explore the potential of novel optical coherence tomography (OCT) imaging technology for clinical diagnosis and cardiovascular research. We believe that quantitative knowledge of protein expression levels contributing to both normal and abnormal pacemaking and conduction could help to formulate future strategies for improved implantable devices as well as tissue engineering approaches in the treatment of heart rhythm disorders associated with the PCS. PUBLIC RELEVANCE: The pacemaking and conduction system (PCS) of the heart is a profoundly complex structure orchestrating orderly contractions of the cardiac chambers by generating and transmitting action potentials at an appropriate rate, conduction velocity, and incorporating a delay between the chambers to allow for proper ventricular filling. Abnormalities in the function of both the sino-atrial and atrio-ventricular nodes of the PCS can cause arrhythmias which are currently treated by electronic pacemakers and/or radio-frequency ablation procedures in up to 500,000 patients annually. The long-term goal of this project is to develop a structure/function framework of the entire human cardiac PCS. During the present funding period we will focus our efforts on the human atrioventricular junction (AVJ). Numerous studies have previously examined the contribution of various isoforms of connexins, ion channels, and receptors to cellular physiology in the cardiac PCS. We, on the other hand, aim to apply a systems physiology approach to examine factors responsible for the stability of normal pacemaking and conduction, abnormal impulse generation and failed conduction, and arrhythmogenesis mediated by the autonomic nervous system. We believe that quantitative knowledge of protein expression levels contributing to both normal and abnormal pacemaking and conduction could help to formulate future strategies for improved implantable devices as well as tissue engineering approaches in the treatment of heart rhythm disorders associated with the PCS.